PRSS22 Mouse

What is mouse PRSS22 and what are its basic characteristics?

Mouse PRSS22 (Protease Serine 22) is a serine protease initially identified as a brain-specific expression gene, though it is primarily expressed in epithelium-rich tissues including the lung and eye. It is also known by several synonyms including Brain-specific serine protease 4 (BSSP-4), Serine protease 26, Tryptase epsilon, and Bssp4 . The full-length mouse PRSS22 protein contains 283 amino acids (positions 33-307) with a molecular mass of approximately 31.1 kDa, though on SDS-PAGE it typically appears at approximately 28-40 kDa due to glycosylation . The protein belongs to the peptidase S1 family and functions as a protease involved in various physiological and pathological processes .

What methods are recommended for detecting PRSS22 expression in mouse tissues?

For detecting PRSS22 expression in mouse tissues, real-time quantitative polymerase chain reaction (RT-qPCR) is commonly employed to measure PRSS22 mRNA levels. The methodology involves:

• Reverse transcription of RNA samples to cDNA using reverse transcriptase cDNA synthesis kits (such as those from Toyobo)

• Quantification via qPCR using SYBR Green Real-time PCR Master Mix on systems like Bio-Rad CFXTM 96 C1000 Real-Time

• Normalization of target gene expression to housekeeping genes such as GAPDH, yielding 2^-ΔCt values for analysis

At the protein level, western blotting and immunohistochemistry can be used with specific anti-PRSS22 antibodies. For protein interaction studies, co-immunoprecipitation (Co-IP) has been successfully employed to confirm protein-protein interactions, as demonstrated in studies of PRSS22's interaction with ANXA1 .

What is the recommended storage and handling protocol for recombinant mouse PRSS22?

For optimal stability and activity of recombinant mouse PRSS22, the following storage and handling protocols are recommended:

• Short-term storage (2-4 weeks): Store at 4°C in the provided buffer

• Long-term storage: Store frozen at -20°C with the addition of a carrier protein (0.1% HSA or BSA) for stability

• Avoid multiple freeze-thaw cycles as these can degrade protein activity and integrity

• The protein is typically supplied as a sterile filtered clear solution

• Standard formulation includes phosphate-buffered saline (pH 7.4) with 10% glycerol at a concentration of 0.5mg/ml

Proper handling ensures the maintenance of >95% purity as determined by SDS-PAGE and preserves the functional characteristics of the recombinant protein for experimental applications .

How does PRSS22 contribute to cancer progression in mouse models?

Studies investigating PRSS22's role in cancer progression have revealed significant insights through mouse models, particularly in breast cancer research. PRSS22 expression is upregulated in breast cancer tissues compared to non-tumorous breast tissues . Mechanistically, research has demonstrated that:

• PRSS22 promotes invasion and metastasis of breast cancer cells both in vitro and in vivo

• Knockdown of PRSS22 inhibits these invasive and metastatic functions

• PRSS22 interacts directly with Annexin A1 (ANXA1), confirmed through protein mass spectrometry, co-immunoprecipitation, and western blot assays

• This interaction leads to the cleavage of ANXA1, generating an N-terminal peptide

• The N-terminal peptide initiates the FPR2/ERK signaling axis, which increases cancer aggressiveness

The transcriptional activation of PRSS22 in cancer models occurs via E2F1, which directly binds to the PRSS22 promoter region as confirmed by dual luciferase assays, bioinformatics analyses, and chromatin immunoprecipitation (ChIP) .

What methodological approaches are recommended for studying PRSS22's enzymatic activity?

To study PRSS22's enzymatic activity as a serine protease, researchers should consider these methodological approaches:

• Substrate specificity assays: Using chromogenic or fluorogenic peptide substrates to determine the cleavage preferences of PRSS22

• Protease activity assays: Measuring the rate of substrate cleavage in the presence of various concentrations of purified recombinant PRSS22

• In vitro cleavage assays: Incubating purified potential substrate proteins (such as ANXA1) with recombinant PRSS22 and analyzing the cleavage products by western blotting or mass spectrometry

• Inhibition studies: Using serine protease inhibitors to confirm the catalytic mechanism of PRSS22

• Site-directed mutagenesis: Creating catalytically inactive mutants by mutating the serine residue in the catalytic triad to study the importance of enzymatic activity in various biological functions

The interaction between PRSS22 and ANXA1 resulting in the generation of an N-terminal peptide provides a specific example of how PRSS22's proteolytic activity can be studied in the context of cancer biology .

What experimental design approaches can be used to effectively study PRSS22 in mouse cancer models?

When designing experiments to study PRSS22 in mouse cancer models, researchers can consider several approaches, including the single mouse experimental design:

• Single mouse experimental design: This approach uses one mouse per treatment group with different patient-derived xenografts, focusing on tumor regression and Event-Free Survival (EFS) as endpoints. This design has been validated to yield similar results as conventional approaches (using 10 mice per group) in ~80% of experiments, and with small allowable differences, the predictive value increases to ~95% .

• Advantages of the single mouse approach:

  • Allows inclusion of 20 models for every one used in conventional testing

  • Facilitates the inclusion of models that better represent genetic/epigenetic diversity of cancer types

  • Requires fewer animals while maintaining statistical power

  • Enables identification of molecular characteristics associated with sensitivity or resistance to treatments

• Implementation considerations:

  • Selection of diverse tumor models with varying characteristics

  • Careful monitoring of tumor growth and regression

  • Correlation of treatment responses with molecular features (e.g., mutations in TP53, 53BP1)

This approach has been validated in prospective studies and can be particularly valuable for studying the role of PRSS22 across different cancer models with diverse genetic backgrounds .

How can researchers effectively analyze PRSS22's role in signaling pathways?

For analyzing PRSS22's role in signaling pathways, particularly in the context of cancer progression, the following methodological approach is recommended:

• Pathway identification:

  • Begin with protein interaction screening via mass spectrometry to identify binding partners

  • Confirm interactions through co-immunoprecipitation and western blot assays

  • Analyze downstream effects using phosphorylation-specific antibodies for key signaling molecules

• Mechanistic validation:

  • Implement gene overexpression and knockdown studies to manipulate PRSS22 levels

  • Use co-overexpression of PRSS22 and its interaction partners (e.g., ANXA1) to observe synergistic effects

  • Employ pathway inhibitors to confirm the involvement of specific signaling components

• Functional readouts:

  • Measure migration and invasion in vitro using transwell assays

  • Assess metastatic potential in vivo using appropriate mouse models

  • Quantify molecular markers of pathway activation (e.g., phosphorylated ERK)

Research has specifically demonstrated that PRSS22 promotes the cleavage of ANXA1, generating an N-terminal peptide that initiates the FPR2/ERK signaling axis, ultimately increasing breast cancer aggressiveness. This provides a template for studying PRSS22's involvement in other potential signaling pathways .

What are the transcriptional regulation mechanisms of PRSS22 in mice and how can they be studied?

The transcriptional regulation of PRSS22 in mice involves several mechanisms that can be studied through specific experimental approaches:

• Identification of transcription factors:

  • Bioinformatic analysis of the PRSS22 promoter region to identify potential transcription factor binding sites

  • Dual luciferase reporter assays to validate promoter activity and response to specific transcription factors

  • Chromatin immunoprecipitation (ChIP) assays to confirm direct binding of transcription factors to the PRSS22 promoter region

• E2F1 as a key regulator:

  • E2F1 has been identified as a direct transcriptional activator of PRSS22

  • E2F1 binds directly to the PRSS22 promoter region and activates its transcription

  • This regulatory mechanism has been confirmed through multiple experimental approaches including ChIP assays

• Experimental methodology:

  • Construction of reporter plasmids containing the PRSS22 promoter region

  • Site-directed mutagenesis of potential transcription factor binding sites

  • Transfection of cells with wild-type and mutant reporter constructs along with expression vectors for transcription factors

  • Measurement of luciferase activity to quantify promoter activation or repression

Understanding the transcriptional regulation of PRSS22 provides insights into how its expression is controlled in normal tissues and dysregulated in disease states such as cancer .

What are common technical challenges when working with recombinant mouse PRSS22?

When working with recombinant mouse PRSS22, researchers may encounter several technical challenges that should be addressed for successful experiments:

• Protein stability issues:

  • PRSS22 may exhibit reduced activity after multiple freeze-thaw cycles

  • Long-term storage without carrier proteins can lead to degradation

  • Solution: Add carrier proteins (0.1% HSA or BSA) for long-term storage and aliquot the protein to avoid repeated freeze-thaw cycles

• Expression system considerations:

  • The choice of expression system (e.g., Sf9 Baculovirus cells) affects glycosylation patterns

  • Molecular weight variations (28-40kDa) on SDS-PAGE due to glycosylation differences

  • Solution: Consider the impact of post-translational modifications on protein function when designing experiments

• Enzymatic activity challenges:

  • Maintaining the catalytic activity of serine proteases during purification and storage

  • Potential for auto-proteolysis

  • Solution: Optimize buffer conditions and consider adding specific protease inhibitors that do not affect PRSS22's catalytic site

• Protein-protein interaction studies:

  • Transient or weak interactions may be difficult to detect

  • Solution: Use crosslinking approaches or proximity-based labeling methods (BioID, APEX) as alternatives to traditional co-immunoprecipitation

How can researchers validate PRSS22 knockdown or knockout effectiveness in mouse models?

Validating PRSS22 knockdown or knockout effectiveness in mouse models requires a multi-level approach to ensure complete characterization:

• Genomic validation:

  • PCR-based genotyping to confirm gene targeting

  • Sequencing of the targeted region to verify the introduced mutations

• Transcriptional validation:

  • RT-qPCR to quantify PRSS22 mRNA levels using validated primers

  • Normalization to housekeeping genes such as GAPDH

  • Calculation of relative expression using the 2^-ΔCt method

• Protein-level validation:

  • Western blotting using specific anti-PRSS22 antibodies

  • Immunohistochemistry or immunofluorescence to examine tissue-specific expression patterns

  • Enzymatic activity assays to confirm functional knockdown/knockout

• Functional validation:

  • Phenotypic analysis focusing on known PRSS22-dependent processes

  • For cancer models, assessment of migration, invasion, and metastatic potential

  • Rescue experiments by re-introducing wild-type PRSS22 to confirm specificity of observed phenotypes

This comprehensive validation approach ensures that any phenotypes observed can be confidently attributed to PRSS22 modulation.

How can mouse PRSS22 research be translated to human cancer studies?

Translating mouse PRSS22 research to human cancer studies requires careful consideration of several factors:

• Comparative analysis:

  • Sequence homology and functional conservation between mouse and human PRSS22

  • Analysis of expression patterns in corresponding normal and cancerous tissues

  • Comparison of protein interaction partners and signaling pathways

• Clinical correlation studies:

  • Analysis of PRSS22 expression in human tumor samples and correlation with clinical outcomes

  • Identification of potential biomarkers associated with PRSS22 activity

  • Investigation of PRSS22's role in specific cancer subtypes where E2F1 dysregulation is common

• Translational methodology:

  • Use of patient-derived xenografts (PDXs) in mouse models to better recapitulate human tumor characteristics

  • Implementation of the single mouse experimental design to expand the diversity of human tumors studied

  • Correlation of treatment responses with molecular features such as TP53 status

• Therapeutic implications:

  • Development of PRSS22 inhibitors as potential therapeutic agents

  • Investigation of combination therapies targeting both PRSS22 and its downstream signaling components

  • Identification of synthetic lethal interactions that could be exploited therapeutically

The established role of PRSS22 in promoting breast cancer aggressiveness through the FPR2/ERK signaling axis provides a foundation for exploring its importance in human cancers and developing targeted interventions .

What emerging technologies can enhance PRSS22 research in mouse models?

Several emerging technologies can significantly enhance PRSS22 research in mouse models:

• CRISPR-Cas9 genome editing:

  • Generation of tissue-specific or inducible PRSS22 knockout mice

  • Introduction of specific mutations to study structure-function relationships

  • Creation of humanized PRSS22 mouse models for better translational research

• Single-cell technologies:

  • Single-cell RNA sequencing to examine PRSS22 expression heterogeneity within tissues

  • Single-cell proteomics to analyze PRSS22 protein levels and post-translational modifications

  • Spatial transcriptomics to map PRSS22 expression within complex tissue architectures

• Advanced imaging techniques:

  • Intravital microscopy to visualize PRSS22-expressing cells in living tissues

  • FRET-based biosensors to monitor PRSS22 activity in real-time

  • Multiplexed immunofluorescence to simultaneously detect PRSS22 and its interaction partners

• Single mouse experimental design:

  • Implementation of this approach allows for testing across a broader range of genetic backgrounds

  • Enables correlation of PRSS22 activity with specific genetic alterations

  • Facilitates identification of biomarkers for PRSS22 sensitivity or resistance

These technologies, combined with traditional approaches, provide a comprehensive toolkit for investigating PRSS22's functions in normal physiology and disease.

What are the key considerations for designing robust PRSS22 mouse studies?

When designing robust PRSS22 mouse studies, researchers should consider the following key factors:

• Experimental design optimization:

  • Consider the single mouse experimental design to increase model diversity

  • Ensure proper controls for genetic background effects

  • Include appropriate sample sizes based on expected effect magnitudes

  • Define clear, measurable endpoints (such as tumor regression and Event-Free Survival)

• Technical considerations:

  • Validate antibodies and reagents specifically for mouse PRSS22

  • Ensure consistent protocols for protein handling and storage

  • Implement multiple methods to confirm knockdown or overexpression efficacy

  • Consider glycosylation and other post-translational modifications

• Mechanistic investigations:

  • Examine both enzymatic and non-enzymatic functions of PRSS22

  • Investigate tissue-specific effects and expression patterns

  • Study interactions with known partners like ANXA1 and explore novel interactions

  • Analyze downstream signaling pathways such as FPR2/ERK

• Translational relevance:

  • Select models that recapitulate human disease features

  • Correlate findings with human patient data when possible

  • Consider potential biomarkers of PRSS22 activity

  • Explore therapeutic implications of PRSS22 modulation